PARENT SESSION
PLATFORM SESSION 6. COMPARATIVE REPRODUCTION AND ENVIRONMENT

Sunday, August 1, 2004
3:00 PM–5:00 PM
Buchanan A203
Chair: Gary Williams Co-Chair: Marcel Amstalden

(46) EXPRESSION OF P450 AROMATASE PROTEIN IN DEVELOPING AND IN SEX-REVERSED GONADS OF THE FROG Rana rugosa.

Nakamura, Masahisa¹, Kato, Takuma¹, Matsui, Kumi¹, ¹ Waseda University, Tokyo, JAPAN

ABSTRACT- In some species of amphibians, gonadal differentiation is very sensitive to steroid hormones. The phenotypic sex of the XX/XY-type frog Rana rugosa can be reversed from female to male by injection of testosterone into tadpoles at st. V-XVI, but little is known about the molecular mechanism of this sex reversal. Thus, we examined the role of P450 aromatase (P450arom) during gonadal differentiation of amphibians to explain the mechanism of the sex differentiation. In this study, we cloned a P450arom cDNA of the frog R. rugosa and analyzed by RT-PCR its expression profile in the developing and in the sex-reversed gonads. The expression of P450arom was observed in the gonad of tadpoles during ovarian differentiation and became much stronger in the developing ovary. However, its expression declined significantly in the ovary of frogs 2 months after metamorphosis, and it was no longer seen in adult ovaries. We also examined by RT-PCR the expression of P450arom and SF-1 (steroidogenic factor-1; the orphan nuclear receptor) in the female-to-male sex-reversed gonad. The level of P450arom mRNA was high in the ovary, but it dropped rapidly after the injection of testosterone. In contrast, no change in the SF-1(also known as Ad4BP) expression was observed. Moreover, we performed immunostaining with an antibody against frog P450arom protein to identify the type(s) of cells expressing P450arom protein. Positive signals were observed in cells around oocytes in the ovary of a frog 1 month after metamorphosis. The positive cells were identified as follicle cells by both light and electron microscopy. The results, taken together, suggest that P450arom protein is synthesized in follicle cells and that P450arom is very much involved in ovarian differentiation in R. rugosa.

KEY WORDS: ovarian development, immunohistochemistry, P450 aromatase, amphibian